Yolanda rodriguez 28nov2011

Extraction and Quantification of Proteins in Syzygium jambos used as

Diabetic Adjuvants in Puerto Rico

Yolanda I. Rodríguez-Rivera BIOL 4990

Mentor: Dr. Jannette Gavillán-Suárez

Proteins

• Proteins are biochemical compounds consisting of polypeptides that facilitate a biological function.

Protein Functions

Ramírez Ramírez, G, Heil, M, Orona Tamayo, D, Adame Álvarez

RM, Ramírez Ramírez, DT, Durán Flores, FD. 2008. ANÁLISIS DE

PROTEÍNAS EN GRÁNULOS Y HOJAS DE Acacia hindsii Y EN

HORMIGAS DE LA REGIÓN DE IRAPUATO . Guanajuato (MX):

Universidad Autónoma de Querétaro (MX). Disponible en:

http://www.uaq.mx/investigacion/difusion/veranos/memorias-

2008/10VeranoRegionCentro/17UAQRamirezRamirez.pdf

Solubility of Proteins

Hydrophilic and Hydrophobic residues on surface

Charged and polar surfaces will have higher solubility

Previous studies A study shows that a protein named ADMc1 in

the seeds of Momordica charantia (cundiamor) has hypoglycemic/anti-hyperglycemic properties.

United States Patent Application Publication, Antidiabetic Protein: Pub.No: US

2011/0046054

Previous studies

• ADMc1 was found highly effective and needed to be administered only once a day to maintain normal blood glucose levels.

• The protein was detected and purified from the seeds of the fruit the plant provides.

• Published work states that the polypeptide-p is made of 166 amino acid residues and said to be of 11 kDa.

Specific aims of this study

To extract proteins on leaves of Syszygium jambos (Pomarrosa del río).

To quantify the proteins by the Lowry method.

Relevance

Determination of proteins that could serve as biomarkers of hypoglycemic/anti-hyperglycemic activities for the reduction of blood glucose in diabetic patients.

Methodology : Extraction of Proteins

3 replicates of the plant

Wash pellet with acetone, 10% TCA and 80% acetone.

Centrifuge.

2 g of of dry pellet is

resuspended in Phenol

Add dense SDS buffer and centrifuge

Separate phases and precipitate Ph phase with methanol and

ammonium acetate.

Recover insoluble

proteins by centrifugation

Wash 3 times with methanolic

ammonium acetate.

Wash with 80% acetone

Quantify proteins in pellet and

supernatant

Methodology: Protein Quantification

BSA Concentration and Calibration

Curve

Add BSA [0, 20, 40, 60, 80, 100 µl] to

these tubes & 100 µl of unknowns

Add 2 ml of Lowry Solution to each one of the test

tubes

Incubate at room temperature for 30

minutes

Add 0.2 ml of diluted Folin-

phenol solution to each tube

Incubate 10 minutes at room

temperature

Vortex each tube immediately

Determine absorbance of each sample at 600 nm

Plot absorbance vs mg BSA to obtain a

calibration curve

Lowry colorimetric reaction

Results

y = 7.7027x + 0.0333 R² = 0.9939

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0 0.005 0.01 0.015 0.02 0.025 0.03 0.035 0.04

Ab

sorb

ance

(6

00

nm

)

BSA Concentration (mg/ml)

BSA Calibration Curve

Results for Syszygium jambos supernatant

• Concentrations of unknowns:

– X1 = 0.33 mg/ml

– X2 = 0.57 mg/ml

– X3 = 0.44 mg/ml

[Protein] = 0.45 mg/ml of supernatant

[Protein] = 0.23 mg/g dry weight

In comparison with:

[Protein] = 0.25 mg/g dry weight (Gómez-Vidal, et.al)

Analysis of Results

• The amount of protein found in this assay is comparable to results in Gómez-Vidal, S, et.al.

• Given that they were found in the supernatant, they could be protein inside the cell.

• The proteins found on the pellet, which are insoluble, are probably structural and could pertain to the cell wall.

Future Work

• Analyze protein in leaves of studied plants and categorize them by their function via 2-dimensional electrophoresis.

• Extract and quantify proteins in leaves of Tapeinochious annasae, Costus speciousus and Tradescantia spathacea.

References

Gómez-Vidal S, Tena M, López-Llorca LV, Salinas J (2007). Protein Extraction from

Phoenix dactylifera L. leaves, a recalcitrant material, for two dimensional electrophoresis. Electrophoresis, 29: 448-456.

Hurkman WJ, Tanaka CK (1986). Solubilization of Plant Membrane Proteins for Analysis by Two-Dimensional Gel Electrophoresis. Plant Physiol., 81: 802-806.

Biology at Davidson [Internet]. 2000. Biology at Davidson in North Carolina. Protein Determination - Lowry Procedure; 2011 Sep 16. Available from: http://www.bio.davidson.edu/people/jowilliamson/Techniques/Protocolweek5.html

Ramírez Ramírez, G, Heil, M, Orona Tamayo, D, Adame Álvarez RM, Ramírez Ramírez, DT, Durán Flores, FD [Internet]. 2008. ANÁLISIS DE PROTEÍNAS EN GRÁNULOS Y HOJAS DE Acacia hindsii Y EN HORMIGAS DE LA REGIÓN DE IRAPUATO . Guanajuato (MX): Universidad Autónoma de Querétaro (MX). Disponible en: http://www.uaq.mx/investigacion/difusion/veranos/memorias-2008/10VeranoRegionCentro/17UAQRamirezRamirez.pdf

Extraction and Quantification of Proteins in Syzygium jambos used as

Diabetic Adjuvants in Puerto Rico

Yolanda I. Rodríguez-Rivera BIOL 4990

Mentor: Dr. Jannette Gavillán-Suárez